defining the normal bacterial flora of the oral cavity
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7/26/2019 Defining the Normal Bacterial Flora of the Oral Cavity
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J Clin Microbiol. 2005 Nov; 43(11): 5721–5732.doi: 10.1128/JCM.43.11.5721-5732.2005
PMC!: PMC1287824
Defining the Normal Bacterial Flora of the Oral Cavity
J"rn #. #$%&1&2&' rc* J. P$%+*r &1&3 ,$r*n N. +o*%&1 n$r l%*n&2 $nd lod . !*ir%+1&3
#+or in6or$+ion #r+icl* no+*% Co9ri+ $nd ,ic*n%* in6or$+ion
i% $r+icl* $% b**n ci+*d b o+*r $r+icl*% in PMC.
ABSTRACT
The oral cavity is comprised of many surfaces, each coated with a
plethora of bacteria, the proverbial bacterial biofilm. Some of these
bacteria have been implicated in oral diseases such as caries and
periodontitis, which are among the most common bacterial infections in
humans. For example, it has been estimated that at least 35% of dentate.S. adults aged 3! to "! years have periodontitis #$. &n addition,
specific oral bacterial species have been implicated in several systemic
diseases, such as bacterial endocarditis #', aspiration pneumonia #(),
osteomyelitis in children #*, preterm low birth weight #), (!, and
cardiovascular disease #(, 3'. Surprisingly, little is +nown about the
microflora of the healthy oral cavity.
y using culture-independent molecular methods, we previously detected
over 5!! species or phylotypes in subgingival plaue of healthy sub/ects
and sub/ects with periodontal diseases #($, necroti0ing ulcerative periodontitis in human immunodeficiency virus-positive sub/ects #(3,
dental plaue in children with rampant caries #3, noma #((, and on the
tongue dorsum of sub/ects with and without halitosis #$5. 1ther
investigators have used similar techniues to determine the bacterial
diversity of saliva #(5, subgingival plaue of a sub/ect with gingivitis
#$), and dentoalveolar abscesses #$!, (". 1ver half of the species
detected have not yet been cultivated. 2ata from these studies have
implicated specific species or phylotypes in a variety of diseases and oral
infections, but still only limited information is available on speciesassociated with health. ecently, 4ager et al. #$" demonstrated
significant differences in the bacterial profiles of '! oral cultivable
species on soft and hard tissues in healthy sub/ects. They also found that
the profiles of the soft tissues were more similar to each other than those
of supragingival and subgingival plaues.
1ur purposes were as follows #i to utili0e culture-independent molecular
techniues to extend our +nowledge on the breadth of bacterial diversity
in the healthy human oral cavity, including not-yet-cultivated phylotypes,
and #ii to determine the site and sub/ect specificity of bacterialcoloni0ation.
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MATERIALS AND METHODS
S!"ect#$
Five sub/ects, representing both genders, ranging in age from (3 to 55
and with no clinical signs of oral mucosal disease were included in the
study. Sub/ects did not suffer from severe halitosis. The periodontia were
healthy in that all periodontal poc+ets were less than 3 mm deep with no
redness or inflammation of the gums. Sub/ects did not have active whitespot lesions or caries on the teeth. 1ur results were consistent with these
clinical observations in that species typically found in caries sub/ects
were not detected #3. The sub/ects had not used antibiotics for the last )
months.
Sam%le collection$
Samples from the following nine sites were analy0ed for each sub/ect
dorsum of the tongue, lateral sides of the tongue, buccal fold, hard palate,
soft palate, labial gingiva and tonsils of soft tissue surfaces, andsupragingival and subgingival plaues from tooth surfaces.
4icrobiological samples of supragingival and subgingival plaue
samples were ta+en with a sterile 6racey curette. 1ther samples were
collected with sterile swab brushes.
Sam%le ly#i#$
7laue samples were directly suspended in 5! 8l of 5! m4 Tris buffer
#p9 :.), $ m4 ;2T<, p9 *, and !.5% Tween (!. 7roteinase = #(!!
8g>ml? oche <pplied Science, &ndianapolis, &@ was added to themixture. The samples were then heated at 55AB for ( h. 7roteinase = was
inactivated by heating at "5AB for 5 min. 2etection of species is
dependent upon obtaining 2@< that can be amplified. Thus, a difficult-
to-lyse bacterium may not be detected. 9owever, by using our lysis
techniue, we were able to detect many hard-to-lyse species, such as
species of Actinomyces and Streptococcus.
Am%lification of &'S rRNA gene# !y (CR an) %rification of(CR %ro)ct#$
The $)S r@< genes were amplified under standardi0ed conditions using
a universal primer set #forward primer, 5C-6<6 <6T TT6 <TD 4T6
6BT B<6-3C? reverse primer, 5C-6<< 66< 66T 6ET BB< BB
6B<-3C #($. 7rimers were synthesi0ed commercially #1peron
Technologies, <lameda, B<. The 7B primers do not necessarily
include all bacterial species. @evertheless, a wide range of phylogenetic
types were obtained in this study and our previous studies by using this
universal primer set #3, $5, ($-(3. 7B was performed in thin-walled
tubes with a 6ene<mp 7B system ":!! #<&, Foster Bity, B<. 1ne
microliter of the lysed sample was added to a reaction mixture #final
volume, 5! 8l containing (! pmol of each primer, '! nmol of
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deoxynucleoside triphosphates, and $ of 7latinum Taq polymerase
#&nvitrogen, San 2iego, B<. &n a hot-start protocol, the samples were
preheated at "5AB for ' min, followed by amplification under the
following conditions denaturation at "5AB for '5 s, annealing at )!AB for
'5 s, and elongation at :(A for $.5 min, with an additional $5 s for each
cycle. < total of 3! cycles were performed? this was followed by a final
elongation step at :(AB for $5 min. The results of the 7B amplification
were examined by electrophoresis in a $% agarose gel. 2@< was stained
with ethidium bromide and visuali0ed under short-wavelength light.Cloning %roce)re#$
Bloning of 7B-amplified 2@< was performed with the T171 T<
cloning +it #&nvitrogen according to the instructions of the manufacturer.
Transformation was done with competent Escherichia coli T17$! cells
provided by the manufacturer. The transformed cells were then plated
onto Guria-ertani agar plates supplemented with +anamycin #5! 8g>ml,
and the plates were incubated overnight at 3:AB. ;ach colony was placed
into '! 8l of $! m4 Tris. Borrect si0es of the inserts were determined ina 7B with an 4$3 #H(! forward primer and an 4$3 reverse primer
#&nvitrogen. 7rior to seuencing of the fragments, the 7B-amplified
$)S r@< fragments were purified and concentrated with 4icrocon $!!
#<micon, edford, 4<, followed by use of the I&<uic+ 7B
purification +it #I&<6;@, alencia, B<.
&'S rRNA gene #e*encing$
7urified 7B-amplified $)S r@< inserts were seuenced using an <&
7rism cycle seuencing +it #ig2ye terminator cycle seuencing +it with
<mpliTaq 2@< polymerase FS, 6ene<mp 7B system ":!!? <&. The
primers used for seuencing have been described previously #($. Iuarter
dye chemistry was used with *! 84 primers and $.5 8l of 7B product in
a final volume of (! 8l. Bycle seuencing was performed with a
6ene<mp 7B system ":!! #<& with (5 cycles of denaturation at
")AB for $! s, annealing at 55A for 5 s, and extension at )!AB for ' min.
The seuencing reactions were run on an <& 3$!! 2@< seuencer
#<&.
&'S rRNA gene #e*encing an) )ata analy#i# of nrecogni+e)in#ert#$
< total of (,5*" clones with an insert of the correct si0e of approximately
$,5!! bases were analy0ed. The number of clones per sub/ect that were
seuenced ranged from '( to )", with an average of 5:.5 and a standard
deviation of :.$. < seuence of approximately 5!! bases was obtained
first to determine identity or approximate phylogenetic position. Full
seuences of about $,5!! bases were obtained by using five to six
additional seuencing primers #$5 for those species deemed novel. For
identification of closest relatives, the seuences of the unrecogni0edinserts were compared to the $)S r@< seuences of over $!,!!!
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microorganisms in our database and over $!!,!!! seuences in the
ibosomal 2atabase 7ro/ect #: and the 6enan+ databases. 1ur cutoff
for species differentiation was (%, or approximately 3! bases for a full
seuence. The similarity matrices were corrected for multiple base
changes at single positions by the method of Ju+es and Bantor #$3.
Similarity matrices were constructed from the aligned seuences by using
only those seuence positions for which data were available for "!% of
the strains tested. 7hylogenetic trees were constructed by the neighbor-
/oining method of Saitou and @ei #('. T;;B1@, a software pac+agefor the 4icrosoft Eindows environment, was used for the construction
and drawing of evolutionary trees #(*. Ee are aware of the potential
creation of $)S r@< chimera molecules assembled during the 7B #$*.
The percentage of chimeric inserts in $)S r@< gene libraries ranged
from $ to $5%. Bhimeric seuences were identified by using the Bhimera
Bhec+ program in the ibosomal 2atabase 7ro/ect, by treeing analysis, or
by base signature analysis. Species identification of chimeras was
obtained, but the seuences were not examined for phylogenetic analysis.
Ncleoti)e #e*ence acce##ion nm!er#$
The complete $)S r@< gene seuences of clones representing novel
phylotypes defined in this study, seuences of +nown species not
previously reported, and published seuences are available for electronic
retrieval from the ;4G, 6enan+, and 22J nucleotide seuence
databases under the accession numbers shown in Fig. Fig.$ $ to to" ".
F&6. $.
acterial profiles of the buccal epithelium of healthy sub/ects.
2istribution and levels of bacterial species>phylotypes among five
sub/ects are shown by the columns of boxes to the right of the tree as
either not detected #clear box, K$5% of ...
F&6. ".
acterial profiles of the subgingival plaue of healthy sub/ects.
2istribution and levels of bacterial species>phylotypes among five
sub/ects are as described for Fig. Fig.$. $. @ovel phylotypes identified in
this study are indicated in bold. 6enan+ ...
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RES,LTS AND DISC,SSION
&t has long been +nown that oral bacteria preferentially coloni0e different
surfaces in the oral cavity as a result of specific adhesins on the bacterial
surface binding to complementary specific receptors on a given oral
surface #$$, $(. &ndeed, the profiles of '! cultivable bacterial species
differed mar+edly on different oral soft tissue surfaces, saliva, and
supragingival and subgingival plaues from healthy sub/ects #$". The
purpose of this study was to define the predominant bacterial flora of the
healthy oral cavity by identifying and comparing the cultivable and thenot-yet-cultivated bacterial species on different soft tissues and supra-
and subgingival plaues.
ased on the analysis of (,5*" $)S r@< clones, the bacterial diversity
of the microflora from nine different sites of five clinically healthy
sub/ects was stri+ingLa total of $'$ different bacterial taxa representing
six different bacterial phyla were detected. The six phyla included
the Firmicutes #previously referred to as the low-6MB gram positives,
such as speciesof Streptococcus, Gemella, Eubacterium, Selenomonas,Veillonella, and
related ones, the Actinobacteria #previously referred to as the high-6MB
gram positives, such as species of Actinomyces, Atopobium, Rothia, and
related ones, the Proteobacteria #e.g., species
of Neisseria, Eikenella, Campylobacter , and related ones,
the Bacteroietes #e.g., species
of Porphyromonas, Pre!otella, Capnocytopha"a, and related ones,
the Fusobacteria #e.g., species of Fusobacterium and #eptotrichia, and
the T4: phylum, for which there are no cultivable representatives. <s
determined in our previous studies using culture-independent molecular
techniues #$5, ($, ((, over )!% of the bacterial flora was represented
by not-yet-cultivated phylotypes. Thirteen new phylotypes #see Fig.
Fig.( ( through through" " were identified for the first time in this study.
&n a comparable ongoing study of caries in permanent teeth, using the
same techniues, we detected ((' species in the analysis of $,(:5 $)S
r@< clones with about )!% as not-yet-cultivable phylotypes #J. <. <as,
S. . 2ardis, <. G. 6riffen, G. @. Sto+es, <. 4. Gee, &. 1lsen, F. ;.
2ewhirst, ;. J. Geys, and . J. 7aster. J. 2ent. es. 84:abstract (*!5,
(!!5. Bonseuently, it is important to identify both the cultivable andnot-yet-cultivated bacterial flora in a given environment before we can
ascribe association to health or disease status. There is no evidence to
suggest that the not-yet-cultivated segment is any less important than the
cultivable segment.
F&6. (.
acterial profiles of the maxillary anterior vestibule of healthy sub/ects.
2istribution and levels of bacterial species>phylotypes among five
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sub/ects are as described for Fig. Fig.$. $. @ovel phylotypes identified in
this study are indicated ...
acterial profiles for each site tested for each sub/ect are shown in nine
phylogenetic trees #see Fig. Fig.$ $ throughthrough". ". &n these
dendrograms, the distribution of bacterial species or phylotypes in each
sub/ect can be observed at a glance. For example, it is clear
that Streptococcus mitis, S$ mitis bv. (, and Gemella hemolysans are the
predominant species of the buccal epithelium #Fig. #Fig.$. $. ;ach of
these species was detected in most or all of the sub/ects and represented
N$5% of the total number of assayed clones #Fig. #Fig.$. $. Similarly, the
predominant bacterial flora for the other eight oral sites was examined. &n
the maxillary anterior vestibule, S$ mitis,Granulicatella spp.,
and Gemella spp. predominated #Fig. #Fig.(. (. 1n the tongue dorsum,
several species of Streptococcus, such as S$ mitis, Streptococcus
australis, Streptococcus parasan"uinis, Streptococcus
sali!arius, Streptococcus sp. clone F7!$5, and Streptococcus sp. clone
F@!5$, Granulicatella aiacens, andVeillonella spp. were the
predominant species #Fig. #Fig.3. 3. 1n the lateral tongue surface #Fig.#Fig.', ', S$ mitis, S$ mitis bv. (, Streptococcus sp. clone
27!!", Streptococcus sp. clone F@!5$, S$ australis, G$ aiacens, G$
hemolysans, and Veillonella spp. predominated. &t is interesting that there
were considerable differences in the bacterial profiles of the tongue
dorsum and the lateral tongue surface. For example, S$ mitis bv. ( was
well represented at the lateral side of the tongue but not detected on the
tongue dorsum. Bonversely,S$parasan"uinis strain *5-*$ was present on
the tongue dorsum, but was absent on the lateral side. This was not
surprising, because these surfaces are +nown to be different inultrastructure and function. For instance, the lateral side of the tongue has
a smooth non+eratini0ed surface, in contrast to the dorsum of the tongue,
which is a +eratini0ed, highly papillated surface with a large surface area
and underlying serous glands. These anatomic differences li+ely influence
the ecology of these habitats and create microbial environmental
differences.
F&6. 3.
acterial profiles of the tongue dorsum of healthy sub/ects. 2istribution
and levels of bacterial species>phylotypes among five sub/ects are as
described for Fig. Fig.$. $. @ovel phylotypes identified in this study are
indicated in bold. 6enan+ ...
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F&6. '.
acterial profiles of the lateral tongue surface of healthy sub/ects.
2istribution and levels of bacterial species>phylotypes among five
sub/ects are as described for Fig. Fig.$. $. @ovel phylotypes identified in
this study are indicated in bold. ...
1n the hard palate, the predominant bacterial species included S$ mitis, S$
mitis biovar (, Streptococcus sp. clone F@!5$, Streptococcus
in%antis, Granulicatella ele"ans, G$ hemolysans, and Neisseria
sub%la!a #Fig. #Fig.5. 5. 1n the soft palate, S$ mitis, other cultivable and
not-yet-cultivable species of Streptococcus, G$ aiacens andG$
hemolysans were predominant #Fig. #Fig.). ). The tonsil bacterial flora
#Fig. #Fig.: : was rather diverseLsome sub/ects
had Pre!otella and Porphyromonas spp. #sub/ects $ and 5, and others
had Firmicutes species #sub/ects (, 3, and '. 1n the tooth surface,
several species of Streptococcus, including Streptococcus sp. clone
;=!'*, S$ san"uinis, and S$ "oronii, and Rothia entocariosa, G$
hemolysans, G$ aiacens, Actinomyces sp. clone G!!*, and Abiotrophia
e%ecti!a were often detected #Fig. #Fig.*. *. Finally, in subgingival plaue, several species of Streptococcus and Gemella were often found
#Fig. #Fig." ".
F&6. 5.
acterial profiles of the hard palate of healthy sub/ects. 2istribution andlevels of bacterial species>phylotypes among five sub/ects are as
described for Fig. Fig.$. $. @ovel phylotypes identified in this study are
indicated in bold. 6enan+ ...
F&6. ).
acterial profiles of the soft palate of healthy sub/ects. 2istribution andlevels of bacterial species>phylotypes among five sub/ects are as
described for Fig. Fig.$. $. @ovel phylotypes identified in this study are
indicated in bold. 6enan+ ...
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F&6. :.
acterial profiles of the tonsils of healthy sub/ects. 2istribution and
levels of bacterial species>phylotypes among five sub/ects are as
described for Fig. Fig.$. $. @ovel phylotypes identified in this study are
indicated in bold. 6enan+ accession ...
F&6. *.
acterial profiles of the tooth surfaces of healthy sub/ects. 2istribution
and levels of bacterial species>phylotypes among five sub/ects are as
described for Fig. Fig.$. $. @ovel phylotypes identified in this study are
indicated in bold. 6enan+ ...
The number of cultivable and not-yet-cultivable species detected in eachsub/ect for each site is also shown in Fig. Fig.$ $ through through". ". For
example, in Fig. Fig.$, $, only four species #S$ mitis, S$mitis bv. (, A$
e%ecti!a, and G$ hemolysans were detected on the buccal epithelium in
sub/ect 5. For convenience, the number of bacterial species detected in
each oral site for each sub/ect is summari0ed in Table Table$. $.
Surprisingly, the highest number of different species was found on the
tonsils in sub/ect $, with (* predominant species? collectively, 5: species
were detected on the tonsils #Table #Table$? $? Fig. Fig.:. :. &n contrast,
the maxillary anterior vestibule had the lowest diversity of the bacterialflora compared with the other sites, with as few as three to nine species
detected among the sub/ects #Table #Table$? $? Fig. Fig.(. (. < common
uestion as+ed is how many bacterial species are present in the oral
cavity of a single individual. The last column of Table Table$ $ indicates
the number of predominant species in each healthy individual from all
nine different sites? thus, the numbers ranged from a low of 3' in sub/ect
' to a high of :( in sub/ect 3.
T<G; $.
@umber of predominant bacterial species per site and sub/ect
1verall, cultivable and not-yet-cultivable species
of Gemella, Granulicatella, Streptococcus, and Veillonellawere
commonly detected in most sites. S$ mitis was the most commonly foundspecies in essentially all sites and sub/ects #Fig. #Fig.$ $ through
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through". ". &n one sub/ect, :"% of the clones identified were S$
mitis #data not shown in figures. @ote that in this study, S$
mitis and Streptococcus pneumoniae have been grouped together. Some
members of the mitis group share more than ""% $)S r@< seuence
similarity, although 2@<-2@< similarity values for the entire
chromosomes are estimated to be less than )!% #$'. oth S$
mitis andStreptococcus oralis have been associated with bacterial
endocarditis, especially in patients with prosthetic valves #". &n addition,
they are now recogni0ed as freuent causes of infection inimmunocompromised patients, particularly immediately after tissue
transplants and in neutropenic cancer patients #3!. G$ aiacens, often
considered an opportunistic pathogen, was also detected at all sites in the
healthy oral cavity. G$ aiacens isolates have been reported from
bacteremia>septicemia in patients with infective endocarditis>atheroma
#33 and in primary bacteremia in patients with neutropenic fever #33. <
high mortality rate for endocarditis by G$ aiacens has been reported #5.
Figure Figure$! $! represents the overall summary of this studyLnamely,
that there are emerging bacterial profiles that help define the healthy oralcavity. <s already discussed, several species, such as S$ mitis and G$
aiacens, were detected in most or all oral sites, whereas several species
were uite site specific. For example, R$
entocariosa, Actinomyces spp., S$ san"uinis, S$"oronii, and A$
e%ecti!a appeared to preferentially coloni0e the teeth, while S$
sali!arius was found mostly on the tongue dorsum. Some species
appeared to have a predilection for soft tissue, e.g., S$ san"uinis and S$
australis did not coloni0e the teeth or subgingival crevice.S$
intermeius preferentially coloni0ed the subgingival plaue in most of thesub/ects but was not detected in most other sites. 1n the other
hand, Neisseria spp. were not found in subgingival plaue but were
present in most other sites. Simonsiella muelleri coloni0ed only the hard
palate. &ndeed, S$ muelleri was initially isolated from the human hard
palate #3(, although it has been isolated from a neonate with dental cyst
and early eruption of teeth #3$. Several Pre!otella species were detected
in most sites, but only in one or two sub/ects. For example, P$
melanino"enica and Pre!otella sp. clone ;!:3 were abundant in seven
out of nine sites of one sub/ect and were detected sporadically in othersub/ects #Fig. #Fig.$!. $!. Pre!otella sp. clone 9F!5! was found in the
maxillary anterior vestibule of one sub/ect, dominating the bacterial flora
as ''% of the clones #Fig. #Fig.( (and and$!. $!. This clone was also
found in lower proportions on the soft palate and tonsils of another
sub/ect #Fig.#Fig.), ), ,:, :, and and$! $!.
F&6. $!.
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Site specificity of predominant bacterial species in the oral cavity. &n
general, bacterial species or phylotypes were selected on the basis of their
detection in multiple sub/ects for a given site. 2istributions of bacterial
species in oral sites among ...
&n conclusion, there is a distinctive bacterial flora in the healthy oral
cavity which is different from that of oral disease. For example, many
species specifically associated with periodontal disease, such
as Porphyromonas "in"i!alis, Tannerella %orsythia, and Treponema
enticola, were not detected in any sites tested. &n addition, the bacterial
flora commonly thought to be involved in dental caries and deep dentin
cavities, represented byStreptococcus
mutans, #actobacillus spp., Bi%iobacterium spp., and Atopobium spp.,
were not detected in supra- and subgingival plaues from clinically
healthy teeth. < more detailed description of bacterial species associated
with oral disease is discussed elsewhere #$:. <s noted in this study on
healthy sub/ects, some species are site specific at one or multiple sites,
while other species are sub/ect specific. <s much as )!% of the species
detected are not presently cultivable. 1verall, there are still more speciesto be discovered, although the number of new species is beginning to
reach saturation.
<s we have previously asserted #($, to rigorously assess the association
of specific species or phylotypes with oral health or disease, it is
necessary to analy0e larger numbers of clinical samples for the levels of
essentially all oral bacteria in well-controlled clinical studies. The
bacterial complexes involved in periodontal disease as defined by
Socrans+y et al. #(: were based on the microbial analyses of $*5
sub/ects representing about $3,!!! plaue samples using 2@< probes to
'! bacterial cultivable species in chec+erboard hybridi0ation assays. Ee
are currently developing 2@< probes for approximately 5!! +nown
bacterial species and novel phylotypes for use in similar studies. These
2@< probes are being developed for use in the chec+erboard
hybridi0ation assay #3, ((, (3 and 2@< microarray formats #S. =.
oches, <. 4. Gee, . J. 7aster, and F. ;. 2ewhirst, J. 2ent.
es. 83:abstract (()3, (!!'. 1ur intent was to first establish the full
bacterial diversity of the oral cavity and then to determine variation and
reproducibility using the microarrays. Bonseuently, it will be relativelyeasy to compare the bacterial composition of a statistically significant
number of samples to more precisely identify those species that are
associated with health and with oral disease from different anatomic sites.
&t is necessary to first define the bacterial flora of the healthy oral cavity
before we can determine the role of oral bacteria in disease.
o +o:
AC-NO.LED/MENTS
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This study was supported by @&9 grant 2;-$$''3 from the @ational
&nstitute of 2ental and Braniofacial esearch and grants from the 2ental
Faculty, niversity of 1slo, @orway.
o +o:
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